Neurobiology of Aging 36 (2015) 1953e1963

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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging

Age-related dysfunctions of the autophagy lysosomal pathway in hippocampal pyramidal neurons under proteasome stress Elena Gavilán a, b,1, Cristina Pintado b, 2, Maria P. Gavilan b, 3, Paula Daza c, Inmaculada Sánchez-Aguayo c, Angélica Castaño a, b, Diego Ruano a, b, * a Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla, Sevilla, Spain b Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain c Departamento de Biología Celular, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 October 2014 Received in revised form 23 February 2015 Accepted 23 February 2015 Available online 28 February 2015

Autophagy plays a key role in the maintenance of cellular homeostasis, and autophagy deregulation gives rise to severe disorders. Many of the signaling pathways regulating autophagy under stress conditions are still poorly understood. Using a model of proteasome stress in rat hippocampus, we have characterized the functional crosstalk between the ubiquitin proteasome system and the autophagy-lysosome pathway, identifying also age-related modifications in the crosstalk between both proteolytic systems. Under proteasome inhibition, both autophagy activation and resolution were efficiently induced in young but not in aged rats, leading to restoration of protein homeostasis only in young pyramidal neurons. Importantly, proteasome stress inhibited glycogen synthase kinase-3b in young but activated in aged rats. This age-related difference could be because of a dysfunction in the signaling pathway of the insulin growth factor-1 under stress situations. Present data highlight the potential role of glycogen synthase kinase-3b in the coordination of both proteolytic systems under stress situation, representing a key molecular target to sort out this deleterious effect. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Autophagy Aging Neurodegeneration Hippocampus GSK-3b

1. Introduction Protein degradation in eukaryotic cells occurs via 2 main pathways: the ubiquitin proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Soluble and short-lived proteins are preferentially degraded by the UPS, whereas both aggregates and long-lived proteins are mainly degraded by the ALP (Ding and Yin, 2008). The 26S proteasome is responsible for catalysis of the ATP-dependent degradation of poly-ubiquitinated proteins (Jung et al., 2009; Rechsteiner and Hill, 2005), being the ubiquitination in K48 the canonical mark for the proteasomal protein degradation (Komander, 2009). Among the 3 types of * Corresponding author at: Instituto de Biomedicina de Sevilla (IBiS)/Hospital Universitario Virgen del Rocío/Universidad de Sevilla, Avenida Manuel Siurot s/n, 41013-Sevilla. Spain. Tel.: þ34 955923052; fax: þ34 954556598. E-mail address: [email protected] (D. Ruano). 1 Present address: Cancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques, Biomedical Research Park, Doctor Aiguader, 88, 08003Barcelona, Spain. 2 Present address: Facultad de Ciencias Ambientales y Bioquímica, Avenida Carlos III s/n, 45071-Toledo, Spain. 3 Present address: Departamento de Señalización Celular, CABIMER, Avd, Americo Vespucio s/n, 41092-Sevilla, Spain. 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2015.02.025

autophagic degradation, macroautophagy (hereinafter referred to as autophagy) is the most important form of autophagy (Mizushima et al., 2008). It involves the formation of a doublemembrane vesicle, called autophagosome, initiated by elongation of a de novoeformed membrane that seals on itself, sequestering cargo inside. Finally, the double-membrane vesicle fuses with lysosomes leading to autophagolysosome formation, where the cargo is degraded. Destination of proteins for proteasome or autophagy degradation is regulated, at least in part, by the Bcl2eassociated athanogen (BAG) proteins. The expressions of BAG1 and BAG3 act as a molecular switch mechanism determining whether proteins are delivered to the proteasome or to the autophagy, respectively (Gamerdinger et al., 2009, 2011; Gavilán et al., 2013). In this sense, BAG3 acts in concert with the ubiquitinbinding protein p62/SQSTM1 to increase autophagic activity. Moreover, p62/SQSTM1 also binds to LC3-II and K63-ubiquitinated proteins, putting together all the elements necessary for the autophagy activation and targeted cargo proteins. Thus, a correct functional crosstalk between both proteolytic systems is crucial to restore protein homeostasis under cellular stress to preserve cell viability. Importantly, it has been shown in vitro that proteasome inhibition activates autophagy, suggesting that both proteolytic

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systems are functionally coupled (Ding et al., 2007; Gavilán et al., 2013). However, the molecular players regulating the relationships between both systems remain to be elucidated. In this sense, autophagy activation induced by proteasome inhibition has been described to be dependent on unfolded protein response (UPR) activation (Ding et al., 2007) or glycogen synthase kinase-3b (GSK3b) inhibition (Gavilán et al., 2013; Parr et al., 2012; Yang et al., 2010). Because the age-related disruption in protein homeostasis makes aged neurons more vulnerable to protein accumulation (Gavilán et al., 2009a), and autophagy disruption is common to some neurodegenerative disorders, an important issue to be elucidated is to know how the 2 major proteolytic systems can interact to each other to preserve protein homeostasis. Here, we demonstrate for the first time in vivo age-related deficiencies in the crosstalk between the UPS and autophagy, with consequences on neuronal protein homeostasis. Also, we provide solid evidence supporting that disruption of the insulin growth factor-1 (IGF-1)/ Akt/GSK3/b-catenin pathway in aged rats could lead to the accumulation of autophagic vacuoles under proteasome stress, highlighting the role of GSK-3b signaling in the maintenance of an effective functional crosstalk between both proteolytic systems. 2. Materials and methods 2.1. Animals Young (3e4 months) and aged (24e26 months) male Wistar rats were provided by the animal care facility of the University of Seville. All experiments were approved by the local ethical committees and complied with international animal welfare guidelines. 2.2. Surgery Young rats (n ¼ 20) and aged rats (n ¼ 20) were processed for surgery and drug injection exactly as previously described (Gavilán et al., 2012). Lactacystin (Sigma-Aldrich, St Louis, MO, USA) was dissolved (10 mg/mL) in a solution of sterilized phosphate-buffered saline (PBS), and 1 mL was injected into both hippocampi. After deep anesthesia with sodium pentobarbital, animals were killed at 6, 14, 24, and 72 hours. Control animals were processed similarly but received 1 mL of sterilized PBS in both hippocampi. 2.3. Sample preparation Both hippocampi were dissected, frozen in liquid N2, and stored at 80  C until use. Hippocampi were homogenized in 700 mL of ice-cold sucrose buffer (0.25 M sucrose, 1 mM EDTA, 10 mM TrisHCl, pH 7.4), supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Three hundred microliters were separated and used for RNA isolation (see subsequently). The remaining homogenate (400 mL) was stored at 80  C until use. Protein concentration was determined by the Lowry method. 2.4. Western blotting Immunoblots were done as previously described (Gavilán et al., 2012). Briefly, proteins were loaded on a 12% or 14% polyacrylamide gel for electrophoresis (sodium dodecyl sulfatepolyacrylamide gel electrophoresis; Bio-Rad, Alcobendas, Spain) and then transferred to a nitrocellulose membrane (Hybond-C Extra, Amersham, Barcelona, Spain). After blocking, membranes were incubated overnight at 4  C with the following primary antibodies: (1) rabbit polyclonal antibodies against Akt, phospho-Akt (S473), Atg7, Beclin1, LC3B, phospho-GSK-3b (S9), and SQSTM1/ p62; K63-linkageespecific poly-ubiquitin (D7A11) (Cell Signaling,

Danvers, MA, USA); ubiquitin (Dako, Glostrup, Denmark); cathepsin B (Santa Cruz Biotech); cathepsin D (ABFrontier Co Ltd); and LAMP-1 (Developmental Studies Hybridoma Bank; University of Iowa); (2) mouse monoclonal antibodies against BAG1 (E-11) (Santa Cruz Biotech); b-actin (Sigma-Aldrich); GSK-3 clone 4G-1E (Millipore, Madrid, Spain); and (3) goat polyclonal antibody against BAG3 (P-17) (Santa Cruz Biotech). Membranes were then incubated with the appropriate secondary antibody (Dako), horseradish peroxidase conjugated, at a dilution of 1/10,000 and developed using the ECL-plus detection method (Amersham) and the ImageQuant LAS 4000 MINI GOLD (GE Healthcare Life Sciences, Barcelona, Spain). For quantification, the optical density of individual bands was analyzed using PCBAS 2.08 software (Raytest Inc, Berlin, Germany) and normalized relative to the optical density of b-actin. 2.5. RNA extraction and reverse transcription For polymerase chain reaction (PCR) analysis, total RNA was extracted using the Tripure Isolation Reagent (Roche, Mannheim, Germany) according to the instructions of the manufacturer. The recovery of RNA was similar in young and aged rats. Reverse transcription was performed using random hexamer primers exactly as previously described (Gavilán et al., 2013). 2.6. Real-time PCR PCR was performed in an ABI Prism 7000 sequence detector (Applied Biosystems, Madrid, Spain) using complementary DNA diluted in sterile water as template. The Bag1, Bag3, Lc3, Atg7, Beclin1, p62/SQSTM1, Tcfeb, and the housekeeper genes were amplified using specific Taqman probes supplied by Applied Biosystems. Threshold cycle (Ct) values were calculated using the software supplied by Applied Biosystems. 2.7. Immunofluorescence and confocal microscopy Young and aged rats were transcardially perfused with 4% paraformaldehyde, and brains were processed as previously described (Gavilán et al., 2007). Sections of 25 mm were cut on a cryostat and mounted on gelatin-coated slides as previously described (Jiménez et al., 2008). Sections from young and aged rats were processed in parallel. They were first permeabilized with 0.5% Triton overnight at room temperature and then incubated with primary antibodies 1 hour at room temperature overnight at 4  C and finally with the appropriate DyLight-conjugated secondary antibodies for 1 hour. Nuclei were counterstained with 40 ,6diamidino-2-phenylindole at a final concentration of 1 ng/mL, after secondary antibody labeling. Control staining included omission of primary antibodies or irrelevant primary antibodies of the same isotype. Then, sections were washed and coverslipped with 0.01 M PBS containing 50% glycerin and 2.5% triethylenediamine and examined under a motorized upright wide-field microscope (Leica DM6000B). Confocal images were captured using a TCS SP5 Confocal Leica laser scanning microscope equipped with a DMI60000 microscope and an HCX PL APO Lambda blue 63 1.4 oil objective at 22  C. Samples were scanned sequentially to avoid crosstalk between fluorochromes, and a maximum projection image was obtained from each series. Image analysis was carried out using the Leica and Adobe Photoshop software. For calculation of granularity profiles, images were processed with MetaMorph Offline 7.1.7.0 using the command granularity. Two sizes of granules were arbitrarily selected: small (0.5e3 mm) and large (3e15 mm) granules.

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2.8. Transmission electron microscopy For ultrastructural analysis, young and aged rats were perfused transcardially with 0.1 M PBS/1% heparin, pH 7.4, followed by 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M PB, pH 7.4. After being removed, the hippocampus was maintained in the same fixative overnight at 4  C, washed several times with PBS, and sectioned into small pieces. The samples were postfixed in 2% osmium tetroxide in 0.1 M PB, dehydrated in acetone series, and embedded in Epon 812 resin. Tissue blocks were cut into semithin (1.5 mm) with a diamond knife in a Leica ultramicrotome (EM UC6), placed on slides, stained with 1% toluidine blue, and explored with the light microscope. Then, selected areas from semithin sections were cut in ultrathin sections, placed on grids, and stained with uranyl acetate and lead citrate before being examined. For immunoelectron microscopy, young and aged rats were perfused transcardially with 0.1 M PBS/1% heparin, pH 7.4, followed by 1% glutaraldehyde-4% paraformaldehyde in 0.1 M PB. Small fragments of hippocampus were dehydrated in alcohol series and embedded in LR White resin. Ultrathin sections were incubated for 2 hours in primary rabbit polyclonal antibodies anti-ubiquitin at 1:50 dilution at room temperature. After washing with PBS, the sections were incubated for 1 hour with goat anti-rabbit immunoglobulin Gecolloidal gold (10 nm). In negative control experiments, the primary antibody was omitted. Sections were examined with an electron microscope Philips CM10 from the Electron Microscopy Service from University of Seville. 2.9. Statistical analyses Data were expressed as mean  standard deviation. For comparison between several groups and ages, we used a multifactor analysis of variance, followed by Bonferroni post hoc multiple comparisons test (Statgraphics plus 3.1; Statpoint Technologies Inc, Warrenton, VA, USA). The significance was set at 95% of confidence. Significant differences are referenced as p < 0.05: “*” with respect to saline-injected animals or “#” with respect to young animals. 3. Results 3.1. BAG1 and BAG3 are differentially regulated in young and aged rats under proteasome stress The BAG1 and BAG3 proteins regulate protein trafficking toward the UPS or the ALP, respectively (Gamerdinger et al., 2009). Because protein homeostasis is disrupted during aging (Koga et al., 2011), we investigated the expression of both proteins in both young and aged rat hippocampus after proteasome inhibition. As shown in Fig. 1A, transcriptional expression of Bag1 was early and significantly increased in young but not in aged rats. On the contrary, transcriptional induction of Bag3 was stronger than Bag1 and affected both young and aged rats (Fig. 1B). Accordingly, the BAG1 isoforms were significantly increased at the protein level in young animals from 14 to 72 hours, whereas aged rats did not modify significantly their expression (Fig. 1C and D). On the other hand, the BAG3 protein was similarly increased in both young and aged rats during the first 24 hours, but it was longer sustained in aged animals (mostly at 72 hours, Fig. 1C). Consequently, the time course for the BAG3/BAG1 protein ratio decreased in young but increased in aged rats in a time-dependent manner (Fig. 1E). Interestingly, the ubiquitination profile was also affected. As shown in Fig. 1F, the amount of K48-linked ubiquitinated proteins, a recognition motif for proteasome degradation (Komander, 2009), was transiently decreased in young animals from 14 to 24 hours, whereas aged rats accumulated K48-linked ubiquitinated proteins at 24 hours but

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practically lacked them at 72 hours. Thus, under proteasome stress, young rats acutely delivered proteins to the ALP, whereas aged rats did so chronically. 3.2. The ALP markers are early upregulated in young but not in aged rats under proteasome stress We next characterized the functional crosstalk between both proteolytic systems in our experimental model. As shown in Fig. 2A and B, basal expression of the specific autophagosome marker ATG8/LC3-II was quite higher in aged than in young animals (38% on average). Proteasome inhibition early decreased the LC3-II content in both young and aged rats (6 hours). Subsequently, young rats displayed a short-term increase followed by a long-term and significant decrease in the LC3-II content (from 24 to 72 hours). By contrast, aged rats showed a time-dependent increase that was significant at 72 hours. Similarly, the expressions of other autophagy-related molecular markers such as ATG7 and Beclin1 were also differentially affected. In young rats, the expression profile of both proteins was similar to LC3-II. However, aged rats showed a different expression pattern for these proteins with no modification during the first 24 hours and a significant decrease (Beclin1) or increase (ATG7) at 72 hours (Fig. 2A, middle and low panels and Fig. 2C and D). These differences could be, at least in part, because of an inefficient transcriptional activation in aged rats. Indeed, quantitative real-time reverse-transcriptase PCR revealed a significant increase in the messenger RNA (mRNA) expression of Lc3, Atg7, and Beclin1 mostly from 14 to 24 hours in young but not in aged rats (Fig. 2E-G). The transcriptional profile matched the behaviors of LC3-II, ATG7, and Beclin1 proteins in young rats mostly from 14 to 24 hours. 3.3. Aggregated ubiquitinated proteins accumulated in aged rats under proteasome stress and codistributed with p62/SQSTM1 and LC3-II proteins We next investigated whether the potential age-related dysfunction in the expression of the autophagic markers could affect protein homeostasis. To address this issue, we analyzed the time course of ubiquitinated proteins accumulation after proteasome inhibition. Because the UPS and the ALP are responsible for degradation of soluble and aggregated proteins, respectively, the study was carried out in parallel in both the supernatant (soluble proteins) and the pellet (aggregated) fractions of young and aged rats. As shown, accumulation of soluble ubiquitinated proteins in young rats was evident during the first 24 hours (Fig. 3A, upper panel). Then, from 24 to 72 hours, the content of aggregated ubiquitinated proteins increased (Fig. 3A, lower panel). By contrast, aged rats accumulated soluble ubiquitinated proteins from 6 to 14 hours, increasing the content of aggregated proteins from 14 to 72 hours (Fig. 3A). Quantification of the pellet/supernatant distribution is shown in Fig. 3B. Interestingly, most of the ubiquitinated proteins in the precipitated fractions were ubiquitinated in K63 (Fig. 3C and compare with Fig. 1F). We also analyzed the expression of the protein p62/SQSTM1, a stress-regulated multiadaptor protein that binds to aggregated polyubiquitinated proteins and LC3, regulating inclusion body formation, and/or autophagy degradation (Bjørkøy et al., 2005; Johansen and Lamark, 2011; Kirkin et al., 2009). Proteasome inhibition led to a sustained and significant transcriptional up-regulation of p62/ SQSTM1 in both young and aged rats, being significantly higher in young animals (Fig. 3D). This was also reflected at the protein level (Fig. 3E, upper panel, and Fig. 3F). Importantly, the supernatant/ pellet distribution of p62/SQSTM1 revealed that in young rats, this protein was preferentially localized as soluble protein in the

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Fig. 1. The Bcl-2eassociated athanogen 1 (BAG1) and BAG3 proteins are differentially expressed in rat hippocampus after proteasome inhibition. (A) Transcriptional expression of Bag1 analyzed by real-time reverse-transcriptase polymerase chain reaction and (B) similar for Bag3. (C) Representative Western blots of the expression of both BAG1 and BAG3 proteins. The 4 BAG1 isoforms were recognized by the primary antibodies: BAG1-L (50 kDa), BAG1-M (46 kDa), BAG1 (36 kDa), and BAG1-S (29 kDa). The experiment was repeated at least 3 times with similar results. (D) Quantification by optical density of Western blots corresponding to BAG1 isoforms. (E) Time course of the BAG3 to BAG1 proteins ratio in young and aged rats after proteasome inhibition. (F) Representative Western blots of the expression of K48-ubiquitinated proteins in young and aged rat hippocampus after proteasome inhibition. Note the decrease in the amount of K48-ubiquitinated proteins in young rats from 14 to 24 hours and also at 72 hours in aged rats. Data are expressed as a percentage  standard deviation (n ¼ 4). *p < 0.05, significant differences compared with control (saline-injected) animals. #p < 0.05, significant differences compared with young animals.

supernatant fractions. The expression of p62/SQSTM1 peaked at 24 hours, preceding the accumulation of ubiquitinated proteins in the pellet fraction, suggesting an active role on autophagosome cargo (Fig. 3E, middle panel). By contrast, the preferential accumulation of p62/SQSTM1 in the precipitated fractions occurred later (probably associated to protein aggregates and/or to autophagosomes). In aged rats, p62/SQSTM1 predominantly accumulated in the pellet fractions from 24 to 72 hours (Fig. 3E, lower panel), in the same way as K63-ubiquitinated proteins and LC3-II did. Indeed, LC3II and ubiquitinated proteins colocalized in the same subcellular fractions (see Supplementary Data and Section 3.6 subsequently). These data indicated that proteasome inhibition severely affected proteostasis in aged rats. 3.4. Proteasome stress-induced transcription factor EB (TFEB) downregulation in aged rats leading to early lysosomal depletion An adequate autophagy resolution implies efficient lysosomal degradation. Thus, we next analyzed the expression of lysosomal

molecular markers. As shown in Fig. 4A, the amount of pro-cathepsin D was quite similar at both ages. However, the content of active cathepsin D was sustained in young rats, whereas it was early abolished in aged rats (Fig. 4B), indicating that processing rather than synthesis of new pro-cathepsin D was disrupted in aged rats. On the other hand, the level of active cathepsin B was early increased in young rats, followed by a time-dependent decrease. By contrast, aged rats accumulated both pro-cathepsin B at 24 hours (Fig. 4A and C). Similarly, the expression of the lysosomal marker LAMP1 was differentially affected (Fig. 4A, lower panel). In both ages, proteasome inhibition lead to an early and significant increase in the content of LAMP1 (Fig. 4D). However, in aged rats, this was followed by a massive depletion of LAMP1 in the first 24 hours, being coincident with cathepsin B accumulation. The same was true for young animals but later (72 hours), being also coincident with pro-cathepsin B accumulation. These data provide strong evidence supporting that lysosomal proteolysis is severely disrupted in aged rats under proteasome stress. Because lysosomal biogenesis and autophagy are coordinated by the master transcription factor EB (Sardiello et al., 2009; Settembre

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Fig. 2. Expression of different macroautophagic markers in young and aged rats after proteasome inhibition. (A) Representative Western blots of LC3/ATG8, ATG7, and Beclin1 after proteasome inhibition in young and aged rats. (BeD) Quantification by optical density of the Western blots corresponding to LC3-II, ATG7, and Beclin1, respectively. Data are expressed as relative units of optical density  standard deviation (SD) (n ¼ 4). (EeG) Quantification of messenger RNA levels of Lc3, Atg7, and Beclin1 by real-time reversetranscriptase polymerase chain reaction. Data are presented as mean  SD (n ¼ 4) of percentage of variation relative to controls. *p < 0.05, significant differences compared with control (saline-injected) animals. #p < 0.05, significant differences compared with young animals.

et al., 2011), we analyzed the transcriptional expression of Tcfeb. Basal expression of Tcfeb was about 70% higher in aged than in young rats (data not shown). However, proteasome inhibition in young rats significantly upregulated Tcfeb transcription, whereas aged rats significantly downregulated its expression (Fig. 4E). 3.5. The IGF-1/Akt/GSK3/b-catenin pathway is activated in young but inactivated in aged rats under proteasome stress We and others have previously demonstrated in vitro that lysosomal biogenesis and autophagy activation are also dependent on GSK-3b signaling (Gavilán et al., 2013; Parr et al., 2012). Thus, we analyzed this relationship in our experimental model. Total amount of GSK-3b and the level of pSer9-GSK-3b were affected in an opposite manner by proteasome inhibition (Fig. 5A). Young rats increased the pSer9-GSK-3b/GSK-3b ratio in a time-dependent manner, whereas aged rats decreased it (Fig. 5B), indicating that proteasome inhibition inactivated GSK-3b in young but not in aged rats. This was supported by the time-dependent accumulation of bcatenin, a target protein of GSK-3b, which exclusively occurred in young rats (Fig. 5A, lower panel). We further analyzed the expression and signaling of IGF-1 as a potential upstream signal that could account for GSK-3b differences. Proteasome inhibition produced an early and robust transcriptional induction of Igf1 in young but not in aged rats (Fig. 5C). This was reflected in a higher and sustained

activation of the IGF-1 receptor, as revealed by the time-dependent increase in the amount of phospho-IGF-1 receptor (Fig. 5D and E) and the concomitant acute Akt activation in young compared with aged rats (Fig. 5F and G). Thus, proteasome inhibition produced a sustained stimulation of the IGF-1/Akt/GSK-3/b-catenin axis in young but not in aged rats. 3.6. Aged pyramidal neurons showed structural features of autophagy dysfunction under proteasome stress We next analyzed at the cellular level whether autophagic degradation affected aged pyramidal neurons. We investigated by immunofluorescence the potential colocalization of both LC3 and ubiquitin. The study was performed at 72 hours when accumulation of both proteins was maximal. Pyramidal neurons accumulated perinuclear punctated structures immunopositive for both LC3 and ubiquitin, supporting colocalization of both proteins (Fig. 6A). These aggregates were higher in size and number in aged compared with young rats (Fig. 6A and B). Because these data could be compatible with both inclusions of protein aggregates and/or nondegraded autophagic vacuoles (see Gavilán et al., 2013), we performed ultrastructural analysis by transition electron microscopy in young and aged lactacystin-treated rats. As shown (Fig. 6C, upper panel), young pyramidal neurons were normal in appearance at 72 hours. Scarce electron-dense structures, which might correspond to

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Fig. 3. Time course of the accumulation of ubiquitinated proteins and of the p62/SQSTM1 expression in young and aged rats after proteasome inhibition. (A) Representative Western blots of soluble (supernatant fraction [SP]) and aggregated (pellet fraction [P]) ubiquitinated proteins. (B) Graphic representation of the aggregated/soluble ratio of ubiquitinated proteins obtained by quantification by optical density of at least 3 Western blots. Data are expressed as mean  standard deviation (SD) of arbitrary units. (C) Representative dot blots of K63-ubiquitinated proteins in the soluble and insoluble fractions of young and aged controls and 72 hours lactacystin-injected rats (n ¼ 4). (D) Analysis by real-time reverse-transcriptase polymerase chain reaction of p62 transcriptional expression. Data are presented as mean  SD (n ¼ 4) of percentage of variation relative to controls. (E) Representative Western blots of p62/SQSTM1 expression in total protein samples (upper panel) and its soluble or aggregated distribution (middle and lower panels). Note the huge increase of p62/SQSTM1 at 24 hours in the soluble fraction of young animals. The experiment was repeated 3 times with the similar results. (F) Quantification by optical density of Western blots corresponding to total p62/SQSTM1. *p < 0.05, significant differences compared with control (saline-injected) animals. #p < 0.05, significant differences compared with young animals.

aggregated protein, could be observed. Moreover, tiny vacuolated structures with heterogeneous content resembling autophagic vacuoles were also found (Fig. 6C, c1 and c3). Interestingly, in some cases, autophagic vacuoles were fused with electron-dense structures (Fig. 6C, c2), suggesting a correct autophagy resolution as indicated in the biochemical data. By contrast, in aged pyramidal neurons, a wide variety of features could be observed. On one hand, big multivacuolated structures heterogeneous in size, which could correspond to lipofuscin-like structures or lipid droplets, were frequently observed (Fig. 6C, left lower panel). Other cells lacked these multivacuolated bodies but displayed large electron-dense cytosolic structures that might correspond to aggregated proteins. Importantly, some of them were surrounded by tubular structures resembling phagophores (Fig. 6C, right lower panel). Finally, multivacuolated bodies and electron-dense cytosolic structures were observed together, and in some cases, electron-dense structures were surrounded by multivacuolated bodies (data not shown). Importantly, none of these structures were observed in young pyramidal neurons. Finally, we analyzed by immunogold labeling

whether cytosolic aggregates were ubiquitinated. Global ubiquitinimmunolabeling was lower in young than in aged neurons (Fig. 6D). In young pyramidal neurons, some small electron-dense structures were faintly labeled for ubiquitin, whereas aged pyramidal neurons showed a plentiful distribution of structures highly immunopositive for ubiquitin. Taken together, these data indicated that agerelated dysfunction in the ALP led to neuronal accumulation of both aggregated proteins and autophagic vacuoles. 4. Discussion Here, we have investigated the functional crosstalk between the UPS and the ALP under stress conditions, looking also for age-related differences. We report data showing that under proteasome stress young rats efficiently activated the ALP. Using the same experimental conditions, we have previously shown that young animals early restored the proteasome chymotryspin-like activity because a rapid proteasome biogenesis (Gavilán et al., 2012). Thus, under proteasome stress, young animals activated autophagy and efficiently induced de

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Fig. 4. Age-related lysosomal dysfunction induced by proteasome inhibition. (A) Representative Western blots showing the expression of lysosomal proteases cathepsins D and B and the lysosomal membrane protein LAMP1 in young and aged rats after proteasome inhibition. The experiment was repeated 3 times with similar results (BeD). Quantification by optical density of Western blots corresponding to active cathepsin D, active cathepsin B, and LAMP1, respectively. (E) Transcriptional analysis by real-time reverse-transcriptase polymerase chain reaction of the transcription factor Tcfeb. Data are presented as mean  standard deviation (n ¼ 4) of percentage of variation relative to controls. *p < 0.05, significant differences compared with control (saline-injected) animals. #p < 0.05, significant differences compared with young animals.

novo proteasome biogenesis. At this time, the activity of the UPS was almost restored, leading to both reduction of autophagy activation (from 14 to 24 hours) and enhancing autophagy resolution (from 24 to 72 hours). In this sense, the expression of p62/SQSTM1 was upregulated later, at the same time that ubiquitinated proteins appeared in the precipitated fractions. The p62/SQSTM1 protein mostly binds to aggregated K63-poly-ubiquitinated proteins and LC3-II, regulating autophagy degradation and/or inclusion body formation (Bjørkøy et al., 2005; Johansen and Lamark, 2011; Kirkin et al., 2009). Collectively, these results supported an efficient functional crosstalk between both proteolytic systems that prevented the accumulation of ubiqutinated proteins and restored proteostasis in young rats. By contrast, aged rats were more dependent on autophagy (Gamerdinger et al., 2009), probably because of the chronic proteasome stress associated to aging (Gavilán et al., 2009a, 2009b; Paz Gavilán et al., 2006). Indeed, proteasome inhibition produced a higher and sustained elevation of the BAG3 to BAG1 ratio in aged rats,

suggesting that proteins were preferentially delivered to the ALP for their degradation. However, none of the autophagic markers were consistently induced (mRNA and protein), indicating a poor autophagic activity. Furthermore, de novo proteasome biogenesis under proteasome stress was lower and occurred later than in young rats (beyond 24 hours) (see Gavilán et al., 2012). Thus, the efficient crosstalk between both proteolytic systems observed in young rats was somehow disrupted in aged animals. The reasons underlying these age-related deficiencies are currently unknown. However, based in previous data, some possibilities should be considered. First, the UPR, a cellular pathway activated under endoplasmic reticulum stress, is linked to autophagy modulation. In particular, the predominant outcome of endoplasmic reticulum stress is the induction, rather than inhibition, of autophagy through the 3 arms of the UPR: IRE1, ATF6, and PERK (Kroemer et al., 2010). In this sense, we have previously showed that both the IRE1 and ATF6 arms were impaired in aged rats under proteasome stress (Gavilán et al., 2009b). Second,

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Fig. 5. The insulin growth factor-1 (IGF-1)/Akt/glycogen synthase kinase-3 (GSK-3)/b-catenin pathway is activated in young but not in aged rats after proteasome inhibition. (A) Representative Western blot showing the time course of both the total amount of GSK-3b and the phospho-Ser9-GSK-3b, in young and old animals after blocking the proteasome activity. (B) Quantification by optical density of the phospho-Ser9-GSK-3b/total GSK-3b ratio in young and aged rats. Data are expressed as mean  standard deviation (SD) of percentage of variation relative to control (n ¼ 4). (C) Transcriptional expression of Igf1 by real-time reverse-transcriptase polymerase chain reaction in young and aged rats after proteasome inhibition. Data are presented as mean  SD of percentage of variation relative to controls (n ¼ 4). (D) Representative Western blot of phosphorylated IGF-1 receptor (IGF-1R) in young and aged rats after proteasome inhibition. The right column corresponds to serum stimulated N2a cells used as control. (E) Quantification by optical density of the Western blots corresponding to phosphorylated IGF-1R. Data are presented as mean  SD of percentage of variation relative to controls (n ¼ 4). (F) Analysis by Western blots of total and phopho-Ser473-Akt in control and at 14 and 24 hours after lactacystin injection in young and aged rats. (G) Quantification by optical density of the phopho-Ser473-Akt/total Akt ratio from Western blots (F). Data are expressed as mean  SD of percentage of variation relative to control (n ¼ 4). *p < 0.05, significant differences compared with control (salineinjected) animals. #p < 0.05, significant differences compared with young animals.

we and others have previously demonstrated in vitro that pharmacological inhibition of GSK-3b induced both autophagy activation and lysosomal biogenesis under proteasome stress (Gavilán et al., 2013; Parr et al., 2012) and restored lysosomal acidification (Avrahami et al., 2013). Here, we demonstrate GSK-3b inhibition under proteasome stress in young but not in aged rats, supporting that GSK-3b might be involve in autophagy activation and resolution also in vivo (see later). Third, infected microglia disrupted neuronal autophagy activation leading to neurodegeneration (Alirezaei et al., 2008). In this line, the presence of activated microglial cells (Gavilán et al., 2007), which alter protein homeostasis in pyramidal neurons, is characteristic of aged hippocampus (Gavilán et al., 2009a; Pintado et al., 2012). Thus, we speculate that several factors such as UPR dysfunction, sustained activity of the GSK-3b, and the presence of activated microglial cells could be age-related factors contributing to the inefficient functional crosstalk between the UPS and the ALP. However, further, studies will be necessary to determine whether all these phenomena are functionally related.

On the other hand, we demonstrate that not only autophagy activation was affected in aged rats but also autophagy resolution was seriously compromised. We provide solid evidence demonstrating that aged rats accumulated LC3-II, p62/SQSTM1, and K63ubiquitinated proteins. Moreover, LC3 and ubiquitin colocalized in aged pyramidal neurons, and they were accumulated in cytosolic structures resembling autophagic vacuoles and aggregateubiquitinated proteins, indicating that autophagy did not compensate for the loss of proteostasis induced by proteasome inhibition. The reasons for these age-related differences are currently unknown. Nevertheless, we point out to lysosomal dysfunction as a potential mechanism underlying this phenomenon. This is based on the fact that LAMP1 was early depleted in aged rats, supporting lysosomal extenuation. Moreover, lysosomal biogenesis is mostly mediated by the master regulator transcription factor EB (TFEB) (Sardiello et al., 2009; Settembre et al., 2011), and we reveal age-dependent decrease in TFBE mRNA expression. Also, processing of cathepsin D and accumulation of both pro-cathepsin B were early observed in

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Fig. 6. LC3 and ubiquitin colocalized in aggregated structures that accumulated in aged pyramidal neurons. (A) Immunofluorescence of LC3 (green) and ubiquitin (red) proteins and colocalization of both (merge) in pyramidal neurons of young and aged animals. 40 ,6-Diamidino-2-phenylindole was used for nuclear staining. Scale bar: 25 mm. (B) Quantification of granularity profiles. Two sizes of granules were arbitrarily selected: small (0.5e3 mm) and large (3e15 mm) granules. (C) Transition electron micrographs of young and aged rats after 72 hours of proteasome inhibition. Young pyramidal neurons (upper panel) were normal in appearance, presenting small and scarce electron-dense structures. In c1, it is shown details of small vacuolated structures with heterogeneous content. In c2, autophagic vacuoles fused with electron-dense structures. In c3, it is shown a large autophagic vacuole. Aged pyramidal neurons (lower panel) often presented multivacuolated structures heterogeneous in size (left lower panel), or big electron-dense cytosolic structures, sometimes surrounded by tubular structures resembling phagophores (right lower panel). Scale bar: 1 mm and 500 nm (c1ec3) (D) Immunogold labeling of ubiquitinated aggregates in young and aged pyramidal neurons. Note the abundant distribution of ubiquitin-immunopositive structures in aged compared with young rats. Most of these structures were frequently surrounded by electron-lucent structures that may correspond to autophagic vacuoles. Note that not all aggregate-like structures were positive for ubiquitin. Scale bar: 1 mm. * Indicates aggregated structures and arrowheads indicates autophagic vacuole like structures. Abbreviation: N, nucleus. Scale bar: 500 nm. #p < 0.05, significant differences compared with young animals. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

aged rats. Cathepsin D is activated by proteolysis in the acidified lysosomes to produce the mature product, and lysosomal acidification is dependent on GSK-3b inhibition (Avrahami et al., 2013; Gieselmann et al., 1985). Thus, the sustained activity of GSK-3b in aged but not in young rats could compromise lysosomal acidification and in turn decrease lysosomal proteolysis (Marchand et al., 2015). This idea is also supported by the presence of lypofuscin-like structures and/or lipid droplets and the accumulation of autophagic vacuoles in aged pyramidal neurons. Collectively, present data indicated that age-related lysosomal dysfunction could result from both lower number of lysosomes and a minor lysosomal function. The age-related dysfunctions in the UPS and the ALP under stress situation could make neuronal cells more vulnerable to the toxicity of protein aggregation. Using the same experimental model, we have previously demonstrated that young rats showed a higher

expression of pro-survival proteins and minimal neurodegeneration, whereas aged rats expressed higher levels of proapoptotic proteins, caspase-3 activation, and neurodegeneration (see Gavilán et al., 2009b). Here, we go further showing that the IGF1/Akt/GSK3/b-catenin axis was stimulated in young but not in aged rats. Importantly, IGF-1 is considered a potent neuroprotective factor in mammals, and its function has been recently shown to be deteriorated in the brain of aged mice (Muller et al., 2012). Moreover, there is compelling evidence supporting the role of b-catenin as a survival element in neurodegenerative diseases (Boonen et al., 2009; Toledo and Inestrosa, 2010). Thus, we speculate that under proteasome stress, the efficient stimulation of the IGF-1/Akt/GSK3/ b-catenin axis could protect neurons against protein toxicity, supporting that autophagy is essential for neuronal survival under proteasome stress (Boland et al., 2008; Gavilán et al., 2009b; Hara

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Fig. 7. Schematic representation of the age-related deficiencies between the ubiquitin proteasome system and the autophagy-lysosome pathway under proteasome stress. Glycogen synthase kinase-3b (GSK-3b) might be acting as a master regulator in the coordination of the functional crosstalk between both proteolytic systems in vivo. We speculate that GSK3b inhibition might coordinate nuclear localization of TFEB, increased number of lysosomes, autophagy activation, lysosomal reacidification, and increased b-catenin stabilization, leading to an effective autophagy activation and resolution that restore protein homeostasis and decreasing neurodegeneration.

et al., 2006; Komatsu et al., 2006). Importantly, it has been described a pro-apoptotic role for Atg7 in lysosomal dysfunction-induced death, in addition to the role on autophagy signaling (Walls et al., 2010). A similar situation is observed here for aged rats under proteasome stress. Moreover, Beclin1 has been described as caspase substrate (Cho et al., 2009). Thus, the increased Atg7 and decreased Beclin1 expression in aged rats at 72 hours could be involved in the caspase-3 induced pyramidal neurons death (Gavilán et al., 2009b), supporting the notion that both Atg7 and Beclin1 might play an important role in regulating both autophagy and apoptosis under proteasome stress in aging. 5. Conclusions We propose that GSK-3b might be acting as a master regulator coordinating the functional crosstalk between both proteolytic systems in vivo. In this sense, GSK-3b inhibition lead to (1) the nuclear localization of TFEB (Marchand et al., 2015; Parr et al., 2012), (2) a higher number of lysosomes (Gavilán et al., 2013; Parr et al., 2012), (3) an increase of autophagy activation (Gavilán et al., 2013; Marchand et al., 2015; Parr et al., 2012; Yang et al., 2010), (4) an increment of lysosomal acidification and proteolytic activity (Avrahami et al., 2013), and (5) an enhancement of b-catenin stabilization and signaling. All these events resulted in a productive stimulation of the ALP, that in addition to an efficient UPR activation (Gavilán et al., 2009b) and a rapid proteasome biogenesis (Gavilán et al., 2012), contributed to reduce neurodegeneration under proteasome stress (Fig. 7). Present findings could have deep implications in the scene of age-related neurodegenerative diseases where proteasome stress is progressively increasing and the GSK3b/b-catenin signaling is impaired, making GSK-3b an attractive pharmacologic target.

Disclosure statement The authors declare no competing financial interests. Acknowledgements We thank Remedios García Navarro, from Departamento de Biología Celular Facultad de Biología de la Universidad de Sevilla, Sandra Macías Benítez, from the Instituto de Biomedicina de Sevilla, and Juan Luis Ribas Salgueiro, from CITIUS, for their technical assistances in transition electron microscopy. This work was supported by grant PI12/00445 (DR) from the Carlos III Health Institute, Spain. EG was supported by a contract from JA. Appendix A. Supplementary Data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2015.02.025. References Alirezaei, M., Kiosses, W.B., Flynn, C.T., Brady, N.R., Fox, H.S., 2008. Disruption of neuronal autophagy by infected microglia results in neurodegeneration. PLoS One 3, e2906. Avrahami, L., Farfara, D., Shaham-Kol, M., Vassar, R., Frenkel, D., Eldar-Finkelman, H., 2013. Inhibition of glycogen synthase kinase-3 ameliorates b-amyloid pathology and restores lysosomal acidification and mammalian target of rapamycin activity in the Alzheimer disease mouse model: in vivo and in vitro studies. J. Biol. Chem. 288, 1295e1306. Bjørkøy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Øvervatn, A., Stenmark, H., Johansen, T., 2005. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin induced cell death. J. Cell Biol. 171, 603e614.

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